High efficiency gene transfer into the embryonic chicken CNS using B-subgroup retroviruses

Attempts to use replication-competent retroviruses to target genes to the chick CNS have met with limited success for injections performed prior to stage 14 using A- or E-subgroup viruses. This study was aimed at improving CNS infection by varying the stage of injection, viral envelope subgroup, viral titer, and the presence or absence of a transgene and/or the polycation polybrene in the inoculum. RCASBP vectors were injected into the neural tube of stages 3-13 embryos and protein expression was determined 9-48 hr later for forebrain, hindbrain, retina, and inner ear. Optimal injection parameters were defined which balanced good survival rates with high levels of transgene expression at early stages. The results demonstrate nearly complete expression of virus-mediated transgenes in neural tissues at stages 15-21 following injection of B-envelope RCASBP with polybrene at stages 7.5-12. This technique can now be applied to study the roles of genes in cell-autonomous events such as cell connectivity, physiology, and differentiation, as well as neural patterning and regional identity.